Phage display-based technologies provide means for cloning, expressing, selecting, and engineering polypeptides with biological functions mediated by their binding to another protein or any other biological target. The iterative process of the affinity-based selection allows the enrichment into relevant clones isolated from large libraries of protein sequences, such as antibodies, epitopes, antigens, bioactive peptides, enzyme inhibitors, enzymes, DNA-binding proteins, isolated protein domains, or ligands for receptors.
In combination with other techniques, phage display technologies, starting from sequences isolated from any kind of nucleic acid-containing material and leading to several types of products (antibodies, enzymes, peptides, etc.), can satisfy a large number of needs for modern biotechnology. Recently, several authors reported not only that functional protein sequences can be obtained using different phage display technologies but also that recombinant phage can be directly used for many applications (such as in diagnostics, for immunization, in proteomics, as antibacterial compounds, in cell transformation, for industrial biotechnology, in nanotechnologies, etc.).
Moreover, the construction of large repertoires of antibody fragments such as variable heavy/light chain heterodimers (or Fabs) and single chain variable regions (or scFv) expressed on the surface of recombinant phage, followed by the affinity-based selection of phage by “panning” on antigens, has been developed as a versatile and rapid method to obtain antibodies having the desired affinity and specificity. This selection process can be subsequently optimized by creating mutant antibody repertoires of the selected phage and sampled for descendents that, for example, bind to antigen under more stringent conditions and with greater affinity.
Phage display technologies take advantage of the small dimension and the adaptability of filamentous phage (such as M13, f1, or Fd), infecting bacterial cells (in particular F-pili bearing Escherichia coli cells) and having highly homologous, single stranded genomes. A large number of vectors, libraries, and display formats have been developed, as reviewed in many recent articles (Sidhu et al., 2000; Benhar, 2001; Sidhu, 2001; Szardenings, 2003; Bradbury and Marks, 2004; Hust and Dubel, 2004; Mancini et al., 2004; Pini et al., 2004; Conrad and Scheller, 2005; Hust and Dubel, 2005; Silacci et al., 2005; Smith et al., 2005), and in books (“Phage display: A practical Approach”, vol. 266, ed. Clackson and Lowman H, Oxford Univ. Press, 2004; “Phage Display: A laboratory Manual”, Burton D R et al., CSHL Press, 2001).
The protein sequences forming the surface of the filamentous phage (called coat proteins) can accommodate and display more or less efficiently heterologous protein sequences that are cloned on their N- or C-terminus forming fusion proteins. Different coat proteins (cp) have been used for this purpose, in particular the minor coat protein (also named as coat protein III/3, g3p, gIIIp, p3, pIII, cpIII, or cp3) and the major coat protein (also named as coat protein VIII/8, g8p, gVIIIp, p8, pVIII, cpVIII, or cp8), but also the other coat proteins cp6, cp7, and cp9 (Gao et al., 1999; Hufton et al., 1999; Kwasnikowski et al., 2005). Depending on the number of copies in which the modified phage protein is present, a distinction can be made between high valency (i.e. when the phage protein is present in a large number of copies, such as for cp8) and low valency (i.e. when the phage protein is present in a few number of copies, such as for cp3, cp6, cp7, or cp9) display, both approaches providing the possibility to select protein sequences binding a specific target.
The fusion of the coat protein with the protein to be displayed can be performed by using either a phage vector or a phagemid vector, providing libraries that can be screened using specific binding agents or targets (O'Connell et al., 2002). In both cases, a coat protein is modified in order to be transcribed and translated into a fusion protein that has a heterologous protein sequence cloned at its N-terminus and exposed by means of a secretion/leader sequence.
When using a phage vector, the DNA coding for the fusion protein is directly cloned into a coat protein of the phage genome, allowing high display levels but with a strong limitation in the size and in the cloning strategy of the heterologous sequence. Several variants to this system have been described, wherein combinations of modified and not modified variants of the same coat protein are present in the same phage vector. Such vectors are defined as type “88”, “33”, or “8+8” (Enshell-Seijffers et al., 2001; Petrenko and Smith, 2004).
When using a phagemid vector, the construct is smaller and comprises sequences triggering the replication during both bacterial and phage cell cycle but only expressing one (or few) of the coat proteins. The phagemid vectors are more easily manipulated by recombinant DNA technology in order to generate libraries of sequences to be expressed on the surface of phage. However, these vectors can provide the complete phage only when the transformed bacterial cells are later infected with a complete phage (the “helper” phage) that supports the correct replication and packaging of the phage, supplying the wild-type version of the coat proteins needed for the reinfection of recombinant phage and the consequent amplification.
Extensive studies have been made on the possibility to optimize the vectors and the sequences to be used for performing phage display screenings, identifying some constraints in using one coat protein rather another (Makowski, 1993) or differences in how heterologous protein sequences are actually displayed by different coat proteins (Iannolo et al., 1995; Weiss and Sidhu, 2000; Weiss et al., 2000; Roth et al., 2002; Li et al., 2003; Held and Sidhu, 2004).
For example, two or more protein sequences having distinct properties (e.g. one binding an antigen and another binding a ligand present on a solid substrate, or different epitope-binding peptides), were displayed on single bifunctional phage (also named in the literature as “dual display” or “double display” phage). Similar phage can be obtained by cloning each sequence in frame with a different coat protein (or a different variant of the same coat protein) into distinct transcription units in the same phagemid vector, assembled in mono- or bicistronic variants, or by double infecting bacterial cells with two phagemid vectors, each one for a specific fusion coat protein (Bonnycastle et al., 1997; Malik and Perham, 1997; Gao et al., 1999; Gao et al., 2002; Chen et al., 2004; WO 98/05344; WO 95/05454, WO 01/25416). While very efficient, the phage display technologies currently available still have room for improvement, and the choice of the coat protein for the display still remains an important issue.
In fact, the efficiency of the system can be substantially affected by the “fitness” of the protein sequence to be expressed, displayed, and screened using one rather than another specific coat protein sequence, a property that cannot be possible to determine in advance. For example, considering the coat proteins more frequently used for this purpose, cp8 seems more appropriate for selecting peptides or low affinity antibodies, due to the binding capacity enhanced by the multivalency of the cp8 system. In contrast, cp3-based display seems more appropriate for selecting high affinity antibodies, given the low number of copies in which the heterologous protein sequences are present on the phage surface. However, given that the affinity of the antibodies in a library is highly variable and impossible to foresee, other factors can affect the display system such as the proteolytic degradation of the cp3-/cp8-based fusion protein in the periplasmic space of E. coli or recombination events eliminating sequences included in the phagemid vector.
Similar problems, that are even more relevant when considering phage-displayed peptides, has been demonstrated in several articles, clearly indicating the need of constructing at least two distinct phage libraries, starting from the same sample containing the DNA coding for the antibody/peptide repertoire to be displayed, for a full exploitation of the potential of this technique. When looking at literature about the use of cp3 and cp8 for displaying and selecting protein sequences, different properties and/or sequences are reported for protein epitopes and peptides (Rousch et al., 1998; Zwick et al., 1998; Adda et al., 1999; Gao and Zhong, 1999; Yip et al., 2001; Al-bukhari et al., 2002; O'Connor K et al., 20051), antibodies (Kretzschmar and Geiser, 1995), or enzymes (Verhaert et al., 1999). Moreover, mixing two phage libraries, each one generated separately with a different phagemid (Jacobsson et al., 2003), or recloning sequences selected using one coat protein into a library displaying another coat protein (Wang et al., 1997) have also been described. Functional protein sequences were also identified using phage displayed libraries wherein random sequences are cloned without a specific orientation, but are actually transcribed and translated directionally by means of regulatory and coding sequences positioned in 5′ and 3′ region to the cloning site, within the vector backbone (Zelenetz and Levy, 1990; van Zonneveld et al., 1995; Stratmann and Kang, 2005).
Several patent applications disclose variants of the phage display technology, such as the combined use of cpVII and cpIX (WO 00/71964), the addition of restriction sites into phage vectors (WO 03/093471, WO 03/91425), the use of bidirectional promoters with heavy and light chain sequences positioned head to head in opposite transcriptional orientations (Den et al., 1999), different combinations encoding sequences arranged for mono- or bi-cistronic expression (Kirsch et al., 2005), the introduction of mutations into coat proteins (WO 02/103012; WO 00/06717), or different approaches for library construction, expression, and screening (WO 98/20036; WO 98/14277; WO 97/35196; WO 97/46251; WO 97/47314; WO 97/09446; WO 03/029456). Alternatively, many documents describe cloning systems based on site-specific recombination for assembling protein sequences into phagemids (WO92/20791, WO95/021914, WO97/020923, WO00/31246, WO96/40714; Tsurushita et al., 1996; Sblattero and Bradbury, 2000).
However, none of these documents discloses how to generate, starting from a library of DNA coding for heterologous proteins and a single phagemid, a single phage library allowing the display of the heterologous proteins fused to either one or the other of two coat proteins.